| 表达siRNA的重组腺相关病毒载体的构建和鉴定 |
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| 引用本文： | 殷俊,李厚达. 表达siRNA的重组腺相关病毒载体的构建和鉴定[J]. 病毒学报, 2010, 26(3) |
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| 作者姓名： | 殷俊 李厚达 |
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| 作者单位： | 扬州大学兽医学院,扬州,225009;扬州大学兽医学院,扬州,225009 |
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| 摘 要： | 腺相关病毒(AAV)载体介导的RNA干扰(RNAi)可在哺乳动物细胞中长期特异性抑制同源基因表达。本研究以AAV Helper-Free System中的转移质粒pAAV-MCS为基础,引入增强型绿色荧光蛋白(EGFP)基因和H1启动子,将该质粒改造为可表达小干扰RNA(siRNA)和EGFP的质粒pAAV-EGFP-H1。该质粒与辅助质粒共转染包装细胞后,获得了具有感染性的重组AAV(rAAV)。以EGFP为靶基因的干扰实验证明:所得rAAV可以产生siRNA并能够特异性抑制EGFP靶基因表达;荧光显微镜观察、FACS分析和Real-time PCR方法均表明重组病毒rAAV-H1-sh EGFP引起EGFP的表达降低大于60%。
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| 关 键 词： | RNA干扰 H1启动子 腺相关病毒 |
Construction and Identification of Recombinant Adeno-associated Virus Expressing siRNA |
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| YIN Jun,LI Hou-da. Construction and Identification of Recombinant Adeno-associated Virus Expressing siRNA[J]. Chinese journal of virology, 2010, 26(3) |
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| Authors: | YIN Jun LI Hou-da |
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| Affiliation: | YIN Jun,LI Hou-da (College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China) |
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| Abstract: | Adeno-associated virus (AAV) mediated RNA interference can be used to inhibit the expression of homologous genes in different mammalian cells. In this study,a transfer plasmid (pAAV-EGFP-H1) containing the H1 promoter and EGFP-expressing cassette was constructed based on the backbone of pAAV-MCS. Using calcium phosphate precipitation method,pAAV-EGFP-H1 was co-transfected into AAV-293 cells with helper plasmids and infective recombinant AAV was generated. EGFP gene was selected as the interfering target. EG... |
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| Keywords: | RNA interference H1 promoter adeno-associated virus |
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